Partial structure of the phylloxin gene from the giant monkey frog, Phyllomedusa bicolor: parallel cloning of precursor cDNA and genomic DNA from lyophilized skin secretion

Phylloxin is a novel prototype antimicrobial peptide from the skin of Phyllomedusa bicolor. Here, we describe parallel identification and sequencing of phylloxin precursor transcript (mRNA) and partial gene structure (genomic DNA) from the same sample of lyophilized skin secretion using our recently-described cloning technique. The open-reading frame of the phylloxin precursor was identical in nucleotide sequence to that previously reported and alignment with the nucleotide sequence derived from genomic DNA indicated the presence of a 175 bp intron located in a near identical position to that found in the dermaseptins. The highly-conserved structural organization of skin secretion peptide genes in P. bicolor can thus be extended to include that encoding phylloxin (plx). These data further reinforce our assertion that application of the described methodology can provide robust genomic/transcriptomic/peptidomic data without the need for specimen sacrifice.

Metastasis-inducing DNA Regulates the Expression of the Osteopontin Gene by Binding the Transcription Factor Tcf-4

Small 1,000-bp fragments of genomic DNA obtained from human malignant breast cancer cell lines when transfected into a benign rat mammary cell line enhance transcription of the osteopontin gene and thereby cause the cells to metastasize in syngeneic rats. To identify the molecular events underlying this process, transient cotransfections of an osteopontin promoter-reporter construct and fragments of one metastasis-inducing DNA (Met-DNA) have identified the active components in the Met-DNA as the binding sites for the T-cell factor (Tcf) family of transcription factors. Incubation of cell extracts with active DNA fragments containing the sequence CAAAG caused retardation of their mobilities on polyacrylamide gels, and Western blotting identified Tcf-4, beta-catenin, and E-cadherin in the relevant DNA complexes in vitro. Transfection of an expression vector for Tcf-4 inhibited the stimulated activity of the osteopontin promoter-reporter construct caused by transiently transfected active fragments of Met-DNA or permanently transfected Met-DNA. This stimulated activity of the osteopontin promoter-reporter construct is accompanied by an increase in endogenous osteopontin mRNA but not in fos or actin mRNAs in the transfected cells. Permanent transfection of the benign rat mammary cell line with a 20-bp fragment from the Met-DNA containing the Tcf recognition sequence CAAAG caused an enhanced permanent production of endogenous osteopontin protein in vitro and induced the cells to metastasize in syngeneic rats in vivo. The corresponding fragment without the CAAAG sequence was without either effect. Therefore, the regulatory effect of the C9-Met-DNA is exerted, at least in part, by a CAAAG sequence that can sequester the endogenous inhibitory Tcf-4 and thereby promote transcription of osteopontin, the direct effector of metastasis in this system.

A panel of single locus minisatellite DNA probes for application to problems in salmonid aquaculture

Eleven minisatellite DNA locus specific probes, isolated from Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) partial genomic DNA libraries, were tested for cross-hybridization to eleven other salmonid species, i.e. sockeye salmon (Oncorhynchus nerka); coho salmon (O. kisutch), chum salmon (O. keta); pink salmon (O. gorbuscha); chinook salmon (O. tshawytscha); rainbow trout (O. mykiss); brook trout (Salvelinus fontinalis); Arctic charr (S. alpinus); grayling (Thymallus thymallus); huchen (Hucho hucho); pollan (Coregonus autumnalis). Simple single locus profiles for each of these species were revealed by, from two to ten SLPs. These markers are likely to be of great value in addressing several problems in aquaculture of these species.

Genomotyping of Pseudomonas putida strains using P-putida KT2440-based high-density DNA microarrays: implications for transcriptomics studies

Pseudomonas putida KT2440 is the only fully sequenced P. putida strain. Thus, for transcriptomics and proteomics studies with other P. putida strains, the P. putida KT2440 genomic database serves as standard reference. The utility of KT2440 whole-genome, high-density oligonucleotide microarrays for transcriptomics studies of other Pseudomonas strains was investigated. To this end, microarray hybridizations were performed with genomic DNAs of subcultures of P. putida KT2440 (DSM6125), the type strain (DSM291(T)), plasmid pWW0-containing KT2440-derivative strain mt-2 (DSM3931), the solvent-tolerant P. putida S12, and several other Pseudomonas strains. Depending on the strain tested, 22 to 99% of all genetic elements were identified in the genomic DNAs. The efficacy of these microarrays to study cellular function was determined for all strains included in the study. The vast majority of DSM6125 genes encoding proteins of primary metabolism and genes involved in the catabolism of aromatic compounds were identified in the genomic DNA of strain S12: a prerequisite for reliable transcriptomics analyses. The genomotypic comparisons between Pseudomonas strains were used to construct highly discriminative phylogenetic relationships. DSM6125 and DSM3931 were indistinguishable and clustered together with strain S12 in a separate group, distinct from DSM291(T). Pseudomonas monteilii (DSM14164) clustered well with P. putida strains.

Experimental setup and first measurement of DNA damage induced along and around an antiproton beam

Radiotherapy employs ionizing radiation to induce lethal DNA lesions in cancer cells while minimizing damage to healthy tissues. Due to their pattern of energy deposition, better therapeutic outcomes can, in theory, be achieved with ions compared to photons. Antiprotons have been proposed to offer a further enhancement due to their annihilation at the end of the path. The work presented here aimed to establish and validate an experimental procedure for the quantification of plasmid and genomic DNA damage resulting from antiproton exposure. Immunocytochemistry was used to assess DNA damage in directly and indirectly exposed human fibroblasts irradiated in both plateau and Bragg peak regions of a 126 MeV antiproton beam at CERN. Cells were stained post irradiation with an anti-gamma-H2AX antibody. Quantification of the gamma-H2AX foci-dose relationship is consistent with a linear increase in the Bragg peak region. A qualitative analysis of the foci detected in the Bragg peak and plateau region indicates significant differences highlighting the different severity of DNA lesions produced along the particle path. Irradiation of desalted plasmid DNA with 5 Gy antiprotons at the Bragg peak resulted in a significant portion of linear plasmid in the resultant solution.

Improving molecular detection of Candida DNA in whole blood:comparison of seven fungal DNA extraction protocols using real-time PCR

The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood.

Genome-wide DNA profiling of marginal zone lymphomas identifies subtype-specific lesions with an impact on the clinical outcome

Marginal zone B-cell lymphomas (MZLs) have been divided into 3 distinct subtypes (extranodal MZLs of mucosa-associated lymphoid tissue [MALT] type, nodal MZLs, and splenic MZLs). Nevertheless, the relationship between the subtypes is still unclear. We performed a comprehensive analysis of genomic DNA copy number changes in a very large series of MZL cases with the aim of addressing this question. Samples from 218 MZL patients (25 nodal, 57 MALT, 134 splenic, and 2 not better specified MZLs) were analyzed with the Affymetrix Human Mapping 250K SNP arrays, and the data combined with matched gene expression in 33 of 218 cases. MALT lymphoma presented significantly more frequently gains at 3p, 6p, 18p, and del(6q23) (TNFAIP3/A20), whereas splenic MZLs was associated with del(7q31), del(8p). Nodal MZLs did not show statistically significant differences compared with MALT lymphoma while lacking the splenic MZLs-related 7q losses. Gains of 3q and 18q were common to all 3 subtypes. del(8p) was often present together with del(17p) (TP53). Although del(17p) did not determine a worse outcome and del(8p) was only of borderline significance, the presence of both deletions had a highly significant negative impact on the outcome of splenic MZLs.

Discovery of DNA hypermethylation using a DHPLC screening strategy

Promoter hypermethylation is recognized as a hallmark of human cancer, in addition to conventional mechanisms of gene inactivation. As such, many new technologies have been developed over the past two decades to uncover novel targets of methylation and decipher complex epigenetic patterns. However, many of these are either labor intensive or provide limited data, confined to oligonucleotide hybridization sequences or enzyme cleavage sites and cannot be easily applied to screening large sets of sequences or samples. We present an application of denaturing high performance liquid chromatography (DHPLC), which relies on bisulfite modification of genomic DNA, for methylation screening. We validated DHPLC as a methylation screening tool using GSTP1, a well known target of methylation in prostate cancer. We developed an in silico approach to identify potential targets of promoter hypermethylation in prostate cancer. Using DHPLC, we screened two of these targets LGALS3 and SMAD4 for methylation. We show that DHPLC has an application as a fast, sensitive, quantitative and cost effective method for screening novel targets or DNA samples for DNA methylation.

Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.

CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.

DNA in Amphibian and Reptile Venom Permits Access to Genomes Without Specimen Sacrifice

Amphibian defensive skin secretions and reptile venoms are rich sources of bioactive peptides with potential pharmacological/pharmaceutical applications. As amphibian and reptile populations are in rapid global decline, our research
group has been developing analytical methods that permit generation of robust molecular data from non-invasive skin secretion samples and venom samples. While previously we have demonstrated that parallel proteome and venom gland
transcriptome analyses can be performed on such samples, here we report the presence of DNA that facilitates the more widely-used applications of gene sequencing, such as molecular phylogenetics, in a non-invasive manner that circumvents specimen sacrifice. From this “surrogate” tissue, we acquired partial 12S and 16S rRNA gene sequences that are presented for illustration purposes. Thus from a single sample of amphibian skin secretion and reptile venom, robust and complementary proteome, transcriptome and genome data can be generated for applications in diverse scientific disciplines.

A comparison of different pre-lysis methods and extraction kits for recovery of Stretococcus agalacticae (Lancefield group B Streptococcus) DNA from whole blood

Sub-optimal recovery of bacterial DNA from whole blood samples can limit the sensitivity of molecular assays to detect pathogenic bacteria. We compared 3 different pre-lysis protocols (none, mechanical pre-lysis and achromopeptidasepre-lysis) and 5 commercially available DNA extraction platforms for direct detection of Group B Streptococcus (GBS) in spiked whole blood samples, without enrichment culture. DNA was extracted using the QIAamp Blood Mini kit (Qiagen), UCP Pathogen Mini kit (Qiagen), QuickGene DNA Whole Blood kit S (Fuji), Speed Xtract Nucleic Acid Kit 200 (Qiagen) and MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Diagnostics Corp). Mechanical pre-lysis increased yields of bacterial genomic DNA by 51.3 fold (95% confidence interval; 31.6–85.1, p < 0.001) and pre-lysis with achromopeptidase by 6.1 fold (95% CI; 4.2–8.9, p < 0.001), compared with no pre-lysis. Differences in yield dueto pre-lysis were 2–3 fold larger than differences in yield between extraction methods. Including a pre-lysis step can improve the limits of detection of GBS using PCR or other molecular methods without need for culture.

Characterisation of a beta-tubulin gene from the liver fluke, Fasciola hepatica.

This study represents the first ß-tubulin sequence from a trematode parasite, namely, the liver fluke, Fasciola hepatica. PCR of genomic DNA showed that at least one ß-tubulin gene from F. hepatica contains no introns. A number of amino acids in the primary sequence of fluke tubulin are different from those described previously in various nematode species and the cestode, Echinococcus multilocularis. ß-Tubulin is an important target for benzimidazole anthelmintics, although (with the exception of triclabendazole) they show limited activity against F. hepatica. The amino acid differences in fluke ß-tubulin are discussed in relation to the selective toxicity of benzimidazoles against helminths and the mechanism of drug resistance.

Deletion of complement factor H-related genes CFHR1 and CFHR3 is associated with atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is associated with defective complement regulation. Disease-associated mutations have been described in the genes encoding the complement regulators complement factor H, membrane cofactor protein, factor B, and factor I. In this study, we show in two independent cohorts of aHUS patients that deletion of two closely related genes, complement factor H-related 1 (CFHR1) and complement factor H-related 3 (CFHR3), increases the risk of aHUS. Amplification analysis and sequencing of genomic DNA of three affected individuals revealed a chromosomal deletion of approximately 84 kb in the RCA gene cluster, resulting in loss of the genes coding for CFHR1 and CFHR3, but leaving the genomic structure of factor H intact. The CFHR1 and CFHR3 genes are flanked by long homologous repeats with long interspersed nuclear elements (retrotransposons) and we suggest that nonallelic homologous recombination between these repeats results in the loss of the two genes. Impaired protection of erythrocytes from complement activation is observed in the serum of aHUS patients deficient in CFHR1 and CFHR3, thus suggesting a regulatory role for CFHR1 and CFHR3 in complement activation. The identification of CFHR1/CFHR3 deficiency in aHUS patients may lead to the design of new diagnostic approaches, such as enhanced testing for these genes.